HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) high glucose supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin (pen/strep, Invitrogen), and non-essential amino acids (NEAA, Invitrogen). Cells were maintained at 37°C and 5% CO2 in a humidified incubator.
Transfections involving nuclease assays were as follows: 0.4×106 cells were transfected with 2μg Cas9 plasmid, 2μg sgRNA and/or 2μg DNA donor plasmid using Lipofectamine 2000 as per the manufacturer’s protocols. Cells were harvested 3 days after transfection and either analyzed by FACS, or for direct assay of genomic cuts the genomic DNA of ~1 × 106 cells was extracted using DNAeasy kit (Qiagen). For these PCR was conducted to amplify the targeting region with genomic DNA derived from the cells and amplicons were deep sequenced by MiSeq Personal Sequencer (Illumina) with coverage >200,000 reads. The sequencing data was analyzed to estimate NHEJ efficiencies.
For transfections involving transcriptional activation assays: 0.4×106 cells were transfected with (1) 2μg Cas9N-VP64 plasmid, 2μg sgRNA and/or 0.25μg of reporter construct; or (2) 2μg Cas9N- plasmid, 2μg MS2-VP64, 2μg sgRNA-2XMS2aptamer and/or 0.25μg of reporter construct. Cells were harvested 24–48hrs post transfection and assayed using FACS or immunofluorescence methods, or their total RNA was extracted and these were subsequently analyzed by RT-PCR. Here standard taqman probes from Invitrogen for REX1, OCT4, SOX2 and NANOG were used, with normalization for each sample performed against GAPDH.
For transfections involving transcriptional activation assays for specificity profile of sgRNA:Cas9 complexes and TALEs: 0.4×106 cells were transfected with (1) 2μg Cas9N-VP64 plasmid, 2μg sgRNA and 0.25μg of reporter library; or (2) 2μg TALE-TF plasmid and 0.25μg of reporter library; or (3) 2μg control-TF plasmid and 0.25μg of reporter library. Cells were harvested 24hrs post transfection (to avoid the stimulation of reporters being in saturation mode). Total RNA extraction was performed using RNAeasy-plus kit (Qiagen), and standard RT-pcr performed using Superscript-III (Invitrogen). Libraries for next-generation sequencing were generated by targeted pcr amplification of the transcript-tags. Full paper
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